Effect of oestrus synchronisation through ovulation delay by vaccination against foot‐and‐mouth disease in Hanwoo (Bos taurus coreanae) cows

Abstract Background In Korean cattle, after foot‐and‐mouth disease (FMD) vaccination, anovulation increases, acute immune response is stimulated. Objective Here, we aimed to improve the fertility rate by ovulation delay caused by the foot‐and‐mouth disease vaccine. Methods 160 cows (control, FMD, FMD+Gn250 and FMD+Gn500 groups, with 40 cows each) were used. We analysed the ovulation delay, ovulation rate, conception rate and acute‐phase immune responses. Results In the group vaccinated only with FMD, the average follicle size was maintained at 12 mm and ovulation was delayed. The ovulation rate of the FMD+Gn500 group (500 μg gonadotropin‐releasing hormone (GnRH) injections 3 days after the FMD vaccination) was the highest at 81.8%. The ovulation rate of the FMD+Gn250 group (250 μg GnRH injections 3 days after FMD vaccination) was 54.5%, and that of the control group (not FMD vaccinated) was 53.3%. The conception rate was 52.5% (19/40) in the control group, 37.5% (15/40) in the FMD+Gn250 group, and 67.5% (27/40) in the FMD+Gn500 group. Analysis of acute‐phase immune response revealed that the plasma contents of haptoglobin and serum amyloid A increased up to 7 days after vaccination against FMD in all the experimental groups, except the control group. Conclusions We concluded that ovulation delay can be employed to improve conception rate after FMD vaccination through a modified ovulation synchronisation method with GnRH.


INTRODUCTION
Many countries across America, Europe and Asia have recently recommended that regular foot-and-mouth disease (FMD) vaccinations on farms is the only way to prevent FMD (Grubman & Baxt, 2004;Kahn et al., 2002;Park et al., 2013;Rodriguez & Grubman, 2009). However, previously, we reported that FMD vaccination is accompanied by side effects such as delayed ovulation, early embryo loss, sperm infertility, decreased milk production, increased acute immune response as well as increased body temperature, leading to miscarriage (Ferreira et al., 2016;Perumal et al., 2013;Yeruham et al., 2001).
Among the various side effects of FMD vaccination, delayed ovulation is associated with fertility rates. Pregnancy through artificial insemination requires semen to be injected into the uterus when an oocyte is ovulated . However, previous studies have confirmed that FMD vaccination disrupts ovulation by delaying it (D. . The mechanism underlying FMD vaccination-induced ovulation delay is in line with Hansen's inflammation pathways by which infection in the mammary gland (Hansen et al., 2004). According to Hansen's study, it was reported that inflammation leads to anovulation by increasing cytokines and prostaglandin F2α (PGF2α). (Hansen et al., 2004;Peter et al., 1989;Skarzynski et al., 2000;Suzuki et al., 2001).
FMD vaccination aggravates the inflammatory response and activates the acute phase response (Hansen et al., 2004;Rodriguez & Grubman, 2009). An increase in acute-phase immune proteins such as haptoglobin and serum amyloid A inhibits the action of PGF2α and gonadotropin-releasing hormone (GnRH), leading to ovulation delay (Hansen et al., 2004;Kujjo et al., 1995;Suzuki et al., 2001). However, although FMD vaccination decreases conception rate due to ovulation delay and early embryo loss, it is essential to prevent FMD in cows (Ferreira et al., 2016;.
Therefore, we aimed to make use of the side effects of FMD vaccination for ovulation synchronisation.
The primary goal of this study was to develop OvSynch, a new synchronisation program by grafting ovulation delay following FMD vaccination using GnRH and PGF2α. The principle of OvSynch is to induce ovulation through GnRH injection on day 0 at a random stage, PGF2α injection on day 7, second GnRH injection on day 9, and artificial insemination on day 10 (Geary et al., 1998;Moreira et al., 2000;Pursley et al., 1998).
Delayed ovulation after immunisation against FMD adversely affects fertility. However, no studies have been conducted on improved fertility rates due to FMDV. Therefore, in this study, the ovulation rate and fertility rate when the GnRH-based OvSynch method is applied after FMDV are comparatively analysed, and a method to improve the decrease in fertility due to FMDV by controlling the amount of GnRH is to be developed.

Animals
For this study, 160 cows (four groups, with 40 cows, each) from the injection on day 9, and artificial insemination on day 10.

F I G U R E 1
Schematic diagram of the experimental design. The diagonal square is the time of FMD vaccination, grey square is the time of i.m. injection, and circle is the time of ovulation test confirmed through transrectal ultrasonography. Blood collection was performed on day -3, 0, 3, 7 and 11.

Plasma collection and concentration of haptoglobin and serum amyloid A(SAA)
Blood samples were collected from all groups on day -3, 0, 3, 7 and 11 (Figure 1). A detailed method for blood sample collection and analysis using a bovine enzyme linked immunosorbent assay (ELISA) kit (haptoglobin, SAA) has been reported by

Statistical analysis
All statistical analyses were performed using GraphPad Prism (version 8.0.1; GraphPad Software Inc., La Jolla, CA, USA). Differences in plasma haptoglobin and SAA concentrations were analysed using two-way analysis of variance (ANOVA) (Tukey's multiple comparisons test), and the conception rate was analysed using the chi-square test. Statistical significance was set at p < 0.05. and FMD+Gn500 group was injected FMD vaccine at day -3 (except for the control group). The control, FMD+Gn250 and FMD+Gn500 groups were injected with 25 mg PGF2α on day 7, injected with 250 μg GnRH on day 9 and artificial insemination was conducted on day 10. Blood samples were collected on days -3, 0, 3, 7 and 11. administered, ovulation delay was confirmed after FMD vaccination, and the ovulation delay was completely resolved on day 11 (Figure 2a).

Ovulation rate and follicle size
Follicle size showed a pattern similar to synchronisation flow ( Figure 2b). The size of the follicles on day 0 was 13.28 ± 0.65 mm in the FMD, 13.82 ± 0.62 mm in the FMD+Gn250 and 13.93 ± 0.75 mm in the FMD+Gn500 groups, markedly higher than that of the control group (9.56 ± 0.90 mm) (p < 0.05) (Figure 2b). On the 7th day, when 25 mg PGF2α was injected, the follicle size was 12.24 ± 0.67 mm in the FMD, 12.55 ± 0.70 mm in the FMD+Gn250 and 13.26 ± 0.64 mm in the FMD+Gn500 groups, again markedly higher than that of the control group value (10.84 ± 0.78 mm) (p < 0.05) (Figure 2b).

Acute phase immune response and conception rate
The analysis of the concentrations of haptoglobin and SAA, representing the acute phase immune response after FMD vaccination, F I G U R E 3 Plasma concentrations of haptoglobin and serum amyloid A depending on the different treatments (n = 160). (a) Plasma haptoglobin and (b) serum amyloid A levels. The black line connected by '○' represents the mean ovulation rate of the control group (NOT vaccinated). The black line connected by '□' represents the mean ovulation rate of the FMD group (Only vaccinated). The black line connected by '△' represents the mean ovulation rate of the FMD+Gn250 group (250 μg GnRH i.m. injection on day 0). The black line connected by '▽' represents the mean ovulation rate of the FMD+Gn500 group (500 μg GnRH i.m. injection on day 0). The FMD, FMD+Gn250 and FMD+Gn500 groups were injected with the FMD vaccine on day -3 (except for the control group). The control, FMD+Gn250 and FMD+Gn500 groups were injected with 25 mg of PGF2α on day 7, injected with 250 μg GnRH on day 9 and artificial insemination on day 10. Blood collection was performed on day -3, 0, 3, 7 and 11. ***Significance level p < 0.001. confirmed that it dramatically increased up to 7 days after FMD vaccination (p < 0.001) (Figure 3). All vaccination groups (FMD, FMD+Gn250 and FMD+Gn500), except the control group, showed increased levels of haptoglobin and SAA after 3 days of FMD vaccination (p < 0.001) (Figure 3). However, at the time of artificial insemination (day 10), haptoglobin and SAA levels recovered to the basal level in all experimental groups (Figure 3). The pregnancy rate in the FMD+Gn500 group was significantly higher than that in the FMD+Gn250 group (p < 0.05) and we observed no adverse effect on the pregnancy rate compared to the control group (Table 1). TA B L E 1 Pregnancy rates and timing following different treatments (n = 120)

Group
No. of pregnant cows Total Pregnancy rates (%)

DISCUSSION
Various oestrus and ovulation synchronisation programs have been developed to improve the fertility rate, including OvSynch of GnRH and PGF2α bases, progesterone-releasing intravaginal devices (PRID), and J-synch methods using oestrogen and progesterone ratio (Bo et al., 2019;Geary et al., 1998;Moreira et al., 2000;Perez-Mora et al., 2020;Pursley et al., 1998). A method of combining PRID and OvSynch was developed to compensate for the shortcomings of OvSync (Geary et al., 1998;Moreira et al., 2000;Pursley et al., 1998). Parity, body condition score, reproductive and nutritional status, and ovulation delay by FMD vaccine in cows are highly associated with the conception rate after artificial insemination and embryo transfer Perez-Mora et al., 2020;Wu & Zan, 2012). Farm animals must be vaccinated to prevent FMD, but farm profits decrease if the conception rate decreases due to side effects such as ovulation delay and miscarriage of pregnant cows (Ferreira et al., 2016;.
In this study, FMD vaccination-induced ovulation delay was designed to be used as a part of ovulation synchronisation, and our research results confirmed the feasibility of this design. The ovulation synchronisation method, which has been improved so far, is a method of increasing the rutting and fertility rate starting at random stage. However, in this study, a unique situation was artificially created through FMD vaccination, and this was applied to ovulation There are no studies applied to ovulation synchronisation using the side effects such as anovulation of the FMD vaccine. Since FMD vaccination combined with OvSynch can achieve both vaccination and improve the conception rate, this method not only prevents cows from suffering from FMD, it also helps to improve the profit of farms through FMD vaccination.
In conclusion, ovulation delay and decreased conception rate are characteristic side effects of FMD vaccination; however, we focused on using these shortcomings to derive positive effects. By administering twice the amount of GnRH at the time of delay owing to FMD vaccination, oestrous synchronisation became more precise. The method of reverse utilising ovulation delay due to FMD vaccines may help improve the conception rate and consequently financially benefit farmers. the reproductive performance in Korean Native Cows for the domestic